Open Journal of Genetics, 2014, 4, 378-384 Published Online September 2014 in SciRes. http://www.scirp.org/journal/ojgen http://dx.doi.org/10.4236/ojgen.2014.45035

How to cite this paper: Thao, N.T.P., Thuy, N.T., Duy, N.K., Chung, D.C., Si, D.M. and Long, L.T. (2014) Phylogenetic Analysis of Baculovirus Isolates from Diseased Insects in Southern Vietnam. Open Journal of Genetics, 4, 378-384. http://dx.doi.org/10.4236/ojgen.2014.45035

Phylogenetic Analysis of Baculovirus Isolates from Diseased Insects in Southern Vietnam Nguyen Thi Phuong Thao1, Nguyen Thi Thuy1, Nguyen Khac Duy1, Doan Chinh Chung2, Do Minh Si2, Le Thanh Long1* 1Institute of Tropical Biology, Vietnam Academy of Science and Technology, Ho Chi Minh City, Vietnam 2University of Science, Ho Chi Minh City, Vietnam Email: *lelong2510@gmail.com Received 8 July 2014; revised 7 August 2014; accepted 28 August 2014

Copyright © 2014 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

Abstract The aim of this study was to investigate the molecular identification and assess the genetic rela-tionship of baculovirus isolated from Southern Vietnam. The diseased insect samples were col-lected from the different fields. The partial sequence of 450 base pairs of lef-8 gene was amplified and sequenced to assess the genetic variations of baculovirus isolates specific for Spodoptera litu-ra, Helicoverpa zea, and Helicoverpa armigera. The sequences alignment demonstrated that Heli-coverpa zea specific isolates exhibited six single nucleotide polymorphic sites. Whereas, twenty five single polymorphic sites were found in Spodoptera litura specific isolates. Thus, Spodoptera litura specific isolates were higher polymorphic than Helicoverpa zea specific isolates. The genetic distance analyses showed that the distance between Vietnamese baculovirus isolates and Group II Alphabaculovirus isolates was lower than other Baculovirus groups. The phylogeny of Vietnamese isolates in relation to other baculovirus isolates was also determined using partial sequences of lef-8 gene. The phylogenetic tree placed all Vietnamese isolates in Group II Alphabaculovirus, where sev-en Vietnamese Helicoverpa zea specific isolates were most closely related to Helicoverpa zea SNPV, fourteen Vietnamese Spodoptera litura specific isolates were located with Spodoptera litura NPV-G2 in one clade and a Vietnamese Helicoverpa armigera isolate was appeared to be closely related to Helicoverpa armigera SNPV-NNg1, Helicoverpa armigera NPV-C1, Helicoverpa armigera NPV-G4.

Keywords Baculovirus, Helicoverpa armigera, Helicoverpa zea, Lef-8 Gene, Phylogeny, Spodoptera litura

*Corresponding author.

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1. Introduction Baculoviruses are a very diverse group of viruses with double-stranded, circular, supercoiled genomes, with siz-es varying from about 80 to over 180 kb and that encode between 90 and 180 genes [1]. The genome is pack-aged in rod-shaped nucleocapsids that are 230 - 385 nm in length and 40 - 60 nm in diameter [2] [3]. In the most well characterized baculoviruses, the virions are present as two types, occluded virions and budded virions. Ba-culoviruses have been reported from a variety of different species of invertebrates. However, the only well do-cumented hosts are Diptera, Hymenoptera, and Lepidoptera. The Baculoviridae family is divided into four ge-nera: Alphabaculovirus (lepidopteran-specific nucleopolyhedroviruses), Betabaculovirus (lepidopteran-specific Granuloviruses), Gammabaculovirus (hymenopteran-specific nucleopolyhedroviruses) and Deltabaculovirus (dipteran-specific nucleopolyhedroviruses) [4]. PCR-base method has been useful tools to identify baculovirus isolates. The late expression factor 8 (lef-8) was a highly conserved DNA region which was used as targets for degenerate PCR primers [5]. The lef-8 gene encodes for a subunit of the baculovirus RNA polymerase, which initiates transcription from late and very late promoters [6]. Lef-8 was previously suitable for studying baculo-virus phylogeny [7].

Vietnam is a tropical country with 1,687,000 ha for fruit, treenut and vegetable crops [8]. These are favorable natural conditions for insect specific baculoviruses. However, no study of distribution and genetic variation of baculovirus in Vietnam have been conducted. In present study, partial lef-8 gene amplification and sequencing was applied to identify the distribution and genetic relationship of baculovirus in Southern Vietnam and be compared to the other baculovirus isolates.

2. Materials and Methods 2.1. Insects and Virus Collection The diseased insects were collected from Ho Chi Minh City, Binh Duong Province, Ninh Thuan Province (Figure 1). Fourteen Spodoptera specific isolates were obtained from cabbage. Seven Helicoverpa zea specific baculovirus isolates were obtained from tomato and corn. One Helicoverpa armigera isolate was obtained from tomato (Table 1).

2.2. DNA Extraction from Diseased Insects DNA extractions of samples of baculovirus infected insects were performed using DNeasy Tissue Kit (69504, Qiagen) according to manuals of the manufacturer. These samples were kept frozen at −20˚C.

2.3. Viral DNA Amplification by PCR The amplification of partial lef-8 gene was performed using degenerate primers [9] (Table 2). PCR was per-formed in a final volume of 50 µl containing 5 µl 10× PCR Buffer (P2192, Sigma), 1.5 mM MgCl2, 200 µM dNTP (D7295, Sigma), 0.05 units/µl Taq DNA Polymerase (D6677, Sigma), 0.5 µM Forward and Reverse Pri-mer. PCR cycle was performed under the following conditions: one cycle of DNA denaturation at 94˚C in 5 min; 40 cycles at 94˚C in 30 s; annealing at 48˚C in 45 s; extension at 72˚C in 45 s; final extension at 72˚C in 10 min. A single band was visualized following electrophoresis of the reaction product in a 1% agarose gel.

2.4. Phylogenetic Analyses PCR products were purified using ExoSAP-IT PCR Clean up kit and used as sequencing templates. The nucleo-tide sequences were determined using the 3730XL DNA Analyzer. The comparison of lef-8 sequences was per-formed for 22 Vietnamese baculovirus isolates and other isolates from Genbank in Table 3 [10]. The lef-8 se-quences were aligned using CLUSTAL W [11]. Tamura & Nei model was used as a genetic distance model. Neighbor-joining method was applied for phylogenetic construction [12]. Bootstrap analyses (using 1000 repli-cations) were used to assess the confidence in branching order.

3. Results and Discussion In this study, the amplification of lef-8 gene was successful for 22 isolates which were collected from various

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Figure 1. Geographical locations of collecting dis-eased insects. 1: Ninh Thuan Province, 2: Binh Duong Province, 3: Ho Chi Minh City.

Table 1. Baculovirus isolates with their host insect, host plant and sources.

No. Isolate Host insect Host plant Collection area

1 Spli-HCM1 Spodoptera litura Cabbage Ho Chi Minh City

2 Spli-HCM2 Spodoptera litura Cabbage Ho Chi Minh City 3 Spli-HCM3 Spodoptera litura Cabbage Ho Chi Minh City 4 Spli-HCM4 Spodoptera litura Cabbage Ho Chi Minh City 5 Spli-NT1 Spodoptera litura Cabbage Ninh Thuan Province 6 Spli-NT2 Spodoptera litura Cabbage Ninh Thuan Province 7 Spli-NT3 Spodoptera litura Cabbage Ninh Thuan Province 8 Spli-NT4 Spodoptera litura Cabbage Ninh Thuan Province 9 Spli-NT5 Spodoptera litura Cabbage Ninh Thuan Province

10 Spli-NT6 Spodoptera litura Cabbage Ninh Thuan Province 11 Spli-NT7 Spodoptera litura Cabbage Ninh Thuan Province 12 Spli-BD1 Spodoptera litura Cabbage Binh Duong Province 13 Spli-BD2 Spodoptera litura Cabbage Binh Duong Province 14 Spli-BD3 Spodoptera litura Cabbage Binh Duong Province 15 Hear-BD Helicoverpa armigera Tomato Binh Duong Province

16 Hz-HCM1 Helicoverpa zea Tomato Ho Chi Minh City

17 Hz-HCM2 Helicoverpa zea Tomato Ho Chi Minh City

18 Hz-HCM3 Helicoverpa zea Tomato Ho Chi Minh City 19 Hz-BD1 Helicoverpa zea Corn Binh Duong Province 20 Hz-BD2 Helicoverpa zea Corn Binh Duong Province

21 Hz-BD3 Helicoverpa zea Corn Binh Duong Province

22 Hz-BD4 Helicoverpa zea Corn Binh Duong Province

Table 2. Primer pairs used for amplification.

Primers Sequencea Size (base pairs)b

L8F2 gtaaaacgacggccagtNNNACNRCNGARGAYCC 450

L8R2 aacagctatgaccatgMMNCCYTTYTGNCCRTG aDegenerate baculovirus primers are in uppercase type. R, A or G; Y, T or C; M, A or C; W, A or T; N, A, C, G, or T; bExpected size of the amplifi-cation product.

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Table 3. Accession numbers of baculovirus isolates.

Genus Name Abbreviation Accession number

Alphabaculovirus Group I

Antheraea pernyi NPV-Z AnpeNPV-Z NC_008035

Anticarsia gemmatalis MNPV-2D AgMNPV-2D NC_008520

Choristoneura fumiferana DEF MNPV CfDEFMNPV NC_005137

Choristoneura fumiferana MNPV CfMNPV NC_004778

Epiphyas postvittana NPV EppoNPV NC_003083

Hyphantria cunea NPV HycuNPV NC_007767

Orgyia pseudotsugata MNPV OpMNPV NC_001875

Alphabaculovirus Group II

Agrotis ipsilon NPV AgipNPV NC_011345

Agrotis segetum NPV AgseNPV NC_007921

Euproctis pseudoconspersa NPV EupsNPV NC_012639

Helicoverpa armigera NPV-C1 HearNPV-C1 NC_003094

Helicoverpa armigera NPV-G4 HearNPV-G4 NC_002654

Helicoverpa armigera MNPV HearMNPV NC_011615

Helicoverpa armigera SNPV-NNg1 HearSNPV-NNg1 NC_011354

Helicoverpa zea SNPV HzSNPV NC_003349

Leucania separate NPV-AH1 LeseNPV-AH1 NC_008348

Lymantria dispar MNPV LdMNPV NC_001973

Lymantria xylina MNPV LyxyMNPV NC_013953

Mamestra configurata NPV-90-2 MacoNPV-90-2 NC_003529

Mamestra configurata NPV-B MacoNPV-B NC_004117

Spodoptera exigua MNPV SeMNPV NC_002169

Spodoptera frugiperda MNPV-3AP2 SfMNPV-3AP2 NC_009011

Spodoptera litura NPV-II SpliNPV-II NC_011616

Spodoptera litura NPV-G2 SpliNPV-G2 NC_003102

Betabaculovirus

Adoxophyes orana GV AdorGV NC_005038

Agrotis segetum GV AgseGV NC_005839

Choristoneura occidentalis GV ChocGV NC_008168

Cryptophlebia leucotreta GV CrleGV NC_005068

Cydia pomonella GV CpGV NC_002816

Helicoverpa armigera GV HearGV NC_010240

Phthorimea operculella GV PhopGV NC_004062

Plutella xylostella GV PlxyGV NC_002593

Spodoptera litura GV-K1 SpliGV NC_009503

Xestia c-nigrum GV XnGV NC_002331

Deltabaculovirus Culex nigripalpus NPV CuniNPV NC_003084

Gammabaculovirus

Neodiprion abietis NPV NeabNPV NC_008252

Neodiprion lecontei NPV NeleNPV NC_005906

Neodiprion sertifer NPV NeseNPV NC_005905

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areas of Southern Vietnam. The genetic polymorphisms among 22 baculoviruse isolates were indentified and analyzed using lef-8 gene obtained from degenerated primers. A fragment of 450 base pairs was amplified. However, a nucleotide sequence of 335 base pairs was used for the analysis due to the higher quality and relia-bility of sequencing after manual edition. These baculoviruse isolates were distributed in 2 main groups includ-ing 14 Spodoptera litura specific isolates and 7 Helicoverpa zea specific isolates. The bases proportion was dif-ferent between two types of isolates. The base proportion for Spodoptera litura specific isolates from Vietnam was 19.2% of thymine, 20.9% of cytosine, 34.6% of adenine and 25.3% of guanine. The base proportion for He-licoverpa zea specific isolates from Vietnam was 18.8% of thymine, 23.8% of cytosine, 36.4% of adenine and 21% of guanine.

The lef-8 alignment exhibited six single polymorphic sites among Helicoverpa zea specific isolates where the Hz-BD1, Hz-BD2, Hz-BD3, Hz-BD4 isolates differed from the remaining isolates by a G to A transition at posi-tion 1993 of lef-8 gene. The Hz-BD1 and Hz-BD3 isolates also exhibited a G to A transition at position 1972 of lef-8 gene. Whereas, the Hz-HCM2 and Hz-BD2 isolates exhibited an A to G transition at position 1908 of lef-8 gene.

The field baculovirus isolates which were collected from various crops and geographic locations commonly expressed genetic variations [13] [14]. In this study, the Vietnamese Helicoverpa zea specific isolates from Ho Chi Minh City were genetically different from Binh Duong Province in the number of variable positions of lef-8 gene. The alignment revealed 3 variable positions of Hz-HCM1, Hz-HCM2 and Hz-HCM3 isolates at position 1895, 1896 and 1908 in lef-8 gene. Conversely, the Hz-BD1, Hz-BD2, Hz-BD3 and HzBD4 isolates exhibited 6 variations at position 1989, 1896, 1908, 1972, 1993 and 2033 in lef-8 gene.

In contrast to Helicoverpa zea specific isolates, the Spodoptera litura specific isolates were highly variable, displaying a total of twenty five single polymorphic sites, suggesting that Spodoptera litura specific isolates were high polymorphic than Helicoverpa zea specific isolates.

The sequences of Spodoptera litura specific isolates and Helicoverpa zea specific isolates were compared to SpliNPV-G2 [15] and HzSNPV [9], respectively. The lef-8 gene of fourteen Spodoptera litura specific isolates were aligned with SpliNPV-G2 and showed the identity greater than 96%. The lef-8 gene of seven Helicoverpa zea specific isolates were aligned with HzSNPV and showed the identity greater than 98%.

The genetic distances with in Spodoptera litura specific isolates and Helicoverpa zea specific isolates were 0.101 ± 0.065 and 0.053 ± 0.022, respectively, suggesting that the Helicoverpa zea specific isolates were lower genetic variable than Spodoptera litura specific isolates. The genetic distance between groups analyses also showed that the distance between Spodoptera litura specific isolates with Group II Alphabaculovirus were higher than Helicoverpa zea (0.234 ± 0.181 and 0.071 ± 0.044, respectively), indicating inter-specific divergence in Spodoptera litura specific isolates. Thus, the partial sequence of the lef-8 gene of 335 bp used in this study could be applied to discriminate Helicoverpa zea specific isolates and Spodoptera litura specific isolates. The genetic distances between Vietnamese isolates were lower than other distances from other groups (Table 4). This result supported for a close genetic relationship between Vietnamese isolates and Group II Alphabaculovirus.

The neighbor-joining tree was applied to assess the phylogenetic relationship of Vietnamese isolates in Bacu-loviridae family using partial lef-8 gene (Figure 2). The phylogram was rooted using sequence of NeseNPV, which was clustered with other isolates from Gammabaculovirus in one clade (NeabNPA isolate and NeleNPV isolate). The other major clades included the Betabaculovirus and Group I Alphabaculovirus with 46% bootstrap value and 67% bootstrap value, respectively. The Group I Alphabaculovirus originated as an ancestral Group II Alphabaculovirus [16]. All Vietnamese isolates belonged to Group II Alphabaculovirus which appeared para-phyletic as a sister clade to Group I [14]. The Vietnamese isolates formed several smaller clades within Group II Alphabaculovirus. Seven Vietnamese Helicoverpa zea specific isolates were located with HzSNPV isolate. The Helicoverpa zea cluster exhibited two groups: one containing 4 isolates from Binh Duong Province (from Hz- BD1 to Hz-BD4) and the other with 3 isolates from Ho Chi Minh City (from Hz-HCM1 to Hz-HCM3). This clade was supported by high bootstrap value of 91% (Figure 2). The Vietnamese Hear-BD isolate was found in Group II and appeared to be closely related to HearSNPVNNg1, HearNPV-C1 and HearNPV-G4 with 52% bootstrap value. Fourteen Vietnamese Spodoptera litura specific isolates were located with SpliNPV-G2 isolate with 78% bootstrap value, suggesting that these isolates have a close genetic relationship to SpliNPV-G2. There were no subheadings among Spodoptera litura specific isolates from Ho Chi Minh City, Ninh Thuan Province and Binh Duong Province.

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Table 4. Matrix of Tamura & Nei genetic distance among baculovirus isolates. Lower triangular matrix values were mean genetic distances, upper triangular matrix values were standard errors.

Spodoptera (Vietnam) Helicoverpa (Vietnam) Alpha_1 Alpha_2 Beta Delta Gamma

Spodoptera (Vietnam) 0.272 0.352 0.181 0.371 0.712 0.720

Helicoverpa (Vietnam) 0.379 0.236 0.044 0.281 0.767 0.560

Alpha_1 0.472 0.343 0.190 0.360 0.524 0.732

Alpha_2 0.234 0.071 0.261 0.180 0.654 0.596

Beta 0.476 0.399 0.412 0.258 0.687 0.550

Delta 0.873 0.915 0.681 0.760 0.709 1.004

Gamma 0.717 0.639 0.683 0.600 0.503 0.934

Figure 2. Phylogenetic tree constructed from partial lef-8 se-quences of baculovirus isolates by the neighbor-joining analysis method. Bootstrap resampling was done 1000 times, and resulting bootstrap values are shown on the corresponding branches.

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Acknowledgements This study was supported by Department of Science and Technology Ho Chi Minh city, Vietnam.

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